Compositions and methods for treating diseases or disorders using extended release nitric oxide releasing solutions

ABSTRACT

The present invention relates to a liquid nitric oxide releasing solution (NORS) comprised of at least one nitric oxide releasing compound and at least one acidifying agent, wherein the NORS provides an extended release of a therapeutically effective amount of nitric oxide gas (gNO). The present invention also relates to a liquid NORS comprised of at least one nitrite compound having a concentration of no greater than about 0.5% w/v and at least one acidifying agent, wherein the NORS releases a therapeutically effective amount of gNO. The present invention also relates to a method for the treatment of a wound in a human, the method comprising administering to the human a liquid NORS comprised of at least one nitric oxide releasing compound and at least one acidifying agent, wherein the NORS provides an extended release of a therapeutically effective amount of gNO. The present invention also relates to a method for the treatment, prevention, or reduction of incidence of a disease or disorder in a human in need thereof, the method comprising administering to the human a liquid NORS comprised of at least one nitrite compound at a concentration of no greater than about 0.5% w/v and at least one acidifying agent, wherein the NORS releases a therapeutically effective amount of gNO.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/643,305, filed Mar. 10, 2015, now issued as U.S. Pat. No. 9,730,956,which claims priority to U.S. Provisional Patent Application No.61/953,053, filed Mar. 14, 2014, each of which is incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

Endogenous NO has been shown to play a critical role in various bodilyfunctions, including the vasodilatation of smooth muscle,neurotransmission, regulation of wound healing and immune responses toinfection such as bactericidal action directed toward various organisms(Moncada et al., 1991, Pharmacol Rev, 43: 109-42; De Groote et al.,1995, Clin Infect Dis, 21(suppl 2): S162-164).

NO is a free-radical which is lipophilic with a small stokes radiusmaking it an excellent signally molecule enabling it to readily crossthe plasma membrane into the cytosol, and is therefore believed to besuitable for treatment of a variety of indications. For example, NO hasbeen demonstrated to play an important role in wound healing throughvasodilatation, angiogenesis, anti-inflammatory and antimicrobial action(Witte et al., 2002, Amer J of Surg, 183: 406-12). It is hypothesizedthat the antimicrobial and cellular messenger regulatory properties ofthis molecule, delivered in an exogenous gaseous form, might easilyenter the wound milieu and be useful in optimizing the healing ofchronic wounds with specific actions directed at reducing bacterialburden, reducing exudate and improving endogenous debridement.

Further, the therapeutic potential of NO donors for cutaneous lesions,as a broad-spectrum antimicrobial seems promising (Fang, 1997, Amer SocClin Invest, 33: 2818-25; Vazquez-Torres et al., 1999, Nitric Oxide andInfection, 475-88). However, to date, this approach has not beenrealized in clinical commercial applications. This may be due to thetoxic side effects of the carrier compounds of solid, liquid, cream, orother non-gaseous NO donors and specifically, the acidic environmentrequired for release of the NO molecule (Omerod et al., 1999, J InvestDermatol, 113: 392-7; Bauer et al., 1998, Wound Repair Regen, 6:569-77). Adequate efficacy also may not have been demonstrated due tobinding of the nitric oxide with other compounds in the preparations.Endogenous approaches such as intracellular nitric oxide synthase (NOS)stimulation and exogenous wound dressings with either NO-donors orsaturated NO-containing solutions have also failed to release consistentsteady-state concentrations of NO (Shabini et al., 1996, Wound RepairRegen, 4: 353-63). Direct exposure to nitric oxide gas has been used(Stenzler, U.S. Pat. Nos. 6,643,2077, 7,892,198, 7,520,1866 and Miller,et al., 2004, J Cutaneous Med Surg 233-238) to treat wound infections,and while effective, requires that the patient be connected to a gascylinder for 8 hours at a time for treatment. As such, these methods arenot suitable or effective in situations when only a very short time isavailable for administration of the molecule.

Weller, and colleagues, describes a system using inorganic nitrite andan organic acid to produce NO on the skin surface (Weller et al., 1998,J Am Acad Dermatol, 38: 559-63). However, they describe the system asmessy, impractical, causing pain in open wounds and possibly causingfurther damage to wounds. Hardwick, et al., refined the system using aselectively permeable membrane between the reactants and the wound. Theyreported that in an in vitro model it was effective at reducingmicrobial load (Hardwick et al., 2011, Clinical Sci, 100: 395-400).While this method for NO formation can be administered through topicalapplication to a lesion or site of infection (Benjamin et al., U.S. Pat.No. 6,709,681), this treatment method is a short duration exposure,requiring multiple reapplications and is unlikely to treat lesions orinfections that are not present at the specific site of application.

Thus, there is a need in the art for simple and effective non-antibioticbased treatments for humans, particularly in instances where the time orwindow available for administration is short, and where the targetedtreatment site is different than the site of administration. The presentinvention addresses these unmet needs in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 depicts a Hathback and Chemiluminescence device.

FIG. 2A depicts the amount of NO detected at 3, 8, and 15 min as well as3 and 4 hours.

FIG. 2B depicts the amount of NO detected at 24 hours. The X scale isTIME (minutes) from start on measuring point (showing pre-measuringamount as 0-0.1 ppm) and Y scale showing amount of NO (measured in ppb).

FIG. 3 is a graph depicting the antibacterial efficacy of NORS againstA. baumanii, methicillin-resistant S. aureus, and E. coli using NORS ofvarying nitrite concentrations (0.07-0.41%) at pH 3.7. Controlswere—saline, nitrites only at 0.41% (pH 6) and saline with reduced pH to3.7. Error bars indicate standard deviation for three experiments with 3repetitions each.

FIG. 4 depicts LA/BHI agar plates, plated with A. baumanii,methicillin-resistant S. aureus, and E. coli following 10 min exposureto NORS.

FIG. 5 is a graph depicting the viability of serotype 1 (stripes) and 6(squares) of M. haemolytica after treatment with 0.41% NORS for 0.5, 1,2, and 5 minutes. A star represents complete kill.

FIG. 6 is a graph depicting the antiviral efficacy of NORS againstInfluenza H1N1 using NORS of varying nitrite concentrations(0.007-0.14%) at pH 3.7. Controls were—saline, nitrites only at 0.14%(pH 6) and saline with reduced pH to 3.7. Error bars indicate standarddeviation for three experiments with 3 repetitions each.

FIGS. 7A-7B are showing 2 photos of the plaque assay plates for A.saline control and NORS at 0.07% strength, B—pH control and NORS at0.14% strength.

FIGS. 8A-8C are graphs depicting the viability of virus using NORS at0.41% and different initial titers for control (triangle), 1 minutetreatment (square) and 10 minutes treatment (circle). Saline was used ascontrol. (A —IBR, B—BRSV, C—PI3).

FIG. 9 is a schematic diagram of the apparatus constructed to test theeffect of the head space gases generated by NORS on the mycelial growth.

FIG. 10 is a graph depicting the antifungal efficacy of NORS againstTrichophyton mentagrophytes (10A) and Trichophyton rubrum (10B) usingNORS of varying nitrite concentrations (0.007-0.07%). Error barsindicate standard deviation for three experiments with 3 repetitionseach. A* represents significant (P<0.05) difference from control.

FIGS. 11A-11B is a chromatogram of head space gases found after 30minutes of NORS exposure. Shown is a chromatogram produced by a GC-MSdemonstrating the constituents of the headspace gas following 30 minexposure of 0.14% NORS. GC-MS method was calibrated to quantify NO, N₂O,and NO₂ levels. A—GC chromatogram for MW=30-molecules detected arelabeled above each peak. B—MS chromatogram with the molecular weightdetected at 5.4 min.

FIG. 12. Antifungal activity of the head space gases produced from NORS.Two line graphs demonstrating 1. Viability count of T. mentagrophytesmycelia (left Y axis) and 2. Nitrite levels in the exposed fungisolution (right Y axis) measured by Griess Reagent™ Both, after beingexposed to gases generated from NORS into the headspace for 2, 4, 8, 16,and 24 hours. Mycelial viability count is shown as squares while nitriteconcentration is shown as triangles. Error bars indicate standarddeviation from triplicates.

FIGS. 13A-13B depicts the incidence of BRDc after 7 and 14 days postarrival to feedlot. FIG. 13A is a graph depicting the percentage of sickanimals in each group. FIG. 13B is a graph depicting the percentage ofsick animals in the treatment/control group out of total sick animals.White=control. Grey=NO treatment.

FIGS. 14A-14C depicts MetHb levels. FIG. 14A is a graph depicting MetHblevels before, 5 minutes, and 30 minutes after treatment for the controlanimals. FIG. 14B is a graph depicting MetHb levels before, 5 minutes,and 30 minutes after treatment for the NO treated animals. FIG. 14C is agraph depicting the average difference in MetHb values 5 minutes and 30minutes post treatment compared to the values measured before treatment.(grey=control, white=NO treatment) all animals tested in each group.

FIGS. 15A-15B depicts the exhaled NO measured by chemiluminescence. FIG.15A is a spectrum depicting the exhaled NO of the control group. FIG.15B is a spectrum depicting the exhaled NO of the treatment group.

FIG. 16 depicts nitrite concentration in samples. Figure is a showingthe difference in nitrite concentration in samples after treatment(concentration in the 5 minute or 30 minutes post-treatment samplesminus the concentration in the pre-treatment samples). (grey=control,white=NO treatment) Error bars indicate standard deviation for allanimals tested in each group.

FIG. 17 comprised of two photos depict the administration of NORS as amist to ferrets.

FIGS. 18A-18B depict the changes in temperature (18A) and viral titre(18B) 1-5 days post viral installation and treatment with either saline(control) or NORS (0.41%) to ferrets.

DETAILED DESCRIPTION

The present invention relates to the unexpected discovery thatadministering a liquid nitric oxide releasing solution (NORS) to asubject provides a mechanism for delivering an effective amount of thegaseous NO (gNO) to one or both of the sites of administration, or to atargeted treatment site that is distal to the administration site. Forexample, administration of a liquid NORS intranasally provides gNOlocally to a subject while allowing for targeted delivery of gNO to adifferent location in the distal airways or the lung of the subject.Further, the present invention relates to the unexpected discovery thatadministration of a liquid NORS provides for the quick delivery of theliquid NORS to the targeted treatment site, followed by an extended andprolonged release of gNO at the treatment site. The present invention isparticularly suited to field or mobile applications, where time andspace are limited. For example, the present invention is well suited foruse with an ambulatory patient or subject whereby a patient's wound canbe covered with a gauze soaked in NORS, covered with a gas impermeablebandage and sent home while they are continued to be treated with gNOfor another 24 hours.

Accordingly, in one aspect of the invention, the NORS provides anextended release of gNO. In another aspect of the invention, the NORS iscomprised of a low concentration of a nitric oxide releasing compoundand/or a low amount of an acidifying agent. The present invention alsoincludes methods for the treatment of a wound in a subject in needthereof. The present invention also includes a method for reducing thepresence of a bacterium, fungi, virus or other pathogen by administeringa NORS. In one embodiment, the solution may be delivered to at least aportion of the upper respiratory tract of a human. In a furtherembodiment, the solution may be instilled on a dressing below a gasimpermeable or semi-impermeable cover. In a further embodiment, thesolution may be in an open container for soaking a limb with aninfection or wound.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value,as such variations are appropriate to perform the disclosed methods.

As used herein, the term “modulate” is meant to refer to any change inbiological state, i.e. increasing, decreasing, and the like.

As used herein, a “therapeutically effective amount” is an amount of atherapeutic composition sufficient to provide a beneficial effect to asubject to which the composition is administered.

The terms “patient,” “subject” and “individual” are interchangeably usedto mean a warm-blooded animal, such as a mammal, suffering from adisease or disorder. It is understood that humans and animals areincluded within the scope of the term “subject,” “subject” or“individual.”

As used herein, the terms “treatment site” and “site of treatment” areused to mean an area, a region or a site on, or inside the body of, asubject, including a tissue, a wound, a lesion, an abscess, includingintact skin. The treatment sites that can be treated by the methods ofthe invention include any area, region or site on the surface of, orinside the body of, a subject that can be exposed to gaseous nitricoxide. By way of nonlimiting examples, regions and sites that can betreated by the methods of the invention include, but are not limited to,external tissues (e.g. skin, etc.), internal tissues (e.g. mucosa,muscle, fascia, etc.), and internal organs (e.g. lungs, liver, etc.). Itshould be understood that many areas, regions and sites that arenormally not amenable to exposure to gaseous nitric oxide can becomeamenable to exposure to gaseous nitric oxide after a wound, such as, forexample, a surgical incision or traumatic laceration, is introduced tothe body of a subject. Moreover, “treatment site” should not beconstrued to include only those areas, regions or sites that exhibitovert evidence of pathology, but rather should also be construed toinclude areas, regions or sites that may be asymptomatic, i.e., that donot contain overt evidence of pathology, but that may be affectednonetheless and that could, in time, exhibit more overt evidence ofpathology. By way of nonlimiting examples, such a site can include atrauma wound, surgical wound, intact tissue or burn, including thosethat have come into contact with, or which is at risk of potentiallycoming into contact with, a pathogen that can colonize or infect thewound, and can be treated, or prophylactically treated, with the devicesand methods of the invention.

“NORS” as used herein may refer to a nitric oxide releasing solution orsubstance.

A “disease” is a state of health of a subject wherein the subject cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe subject's health continues to deteriorate.

In contrast, a “disorder” in a subject is a state of health in which thesubject is able to maintain homeostasis, but in which the subject'sstate of health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the subject's state of health.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

The term “treat” or “treatment,” as used herein, refers to thealleviation (i.e., “diminution”) and/or the elimination of a sign orsymptom or a source of a sign or symptom of a disease or disorder. Byway of several non-limiting examples, a symptom of a bacterial infectioncan be treated by alleviating a symptom of that disorder. A symptom of abacterial infection can also be treated by altogether eliminating asymptom of that disorder. A bacterial infection or colonization can betreated by alleviating the source, or “cause,” of that disorder. Abacterial infection or colonization can also be treated by eliminatingthe source of that disorder.

As used herein, an “antibiotic-resistant bacterium,” is a bacterium thatis a member of a species of bacteria that has historically exhibitedgreater susceptibility to one or more particular antibiotic agents thanthe antibiotic-resistant member bacterium presently exhibits.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

As mentioned previously, the present invention relates to systems andmethods for administering a liquid NORS to a subject as a vehicle forreleasing an effective amount of gNO to the site of administrationand/or to a targeted treatment site that is distal to the administrationsite. Administration of the liquid NORS provides for the quick deliveryof the liquid NORS to the targeted treatment site, followed by anextended and prolonged release of gNO at the treatment site.

The present invention provides a number of advantages over currentlyused NO treatments. For example, as presented herein, it has beenunexpectedly found that the NORS of the present invention are capable ofreleasing a therapeutically effective amount of NO for an extendedperiod of time while using a lower amount of one or both of a nitritecomponent and an acidifying agent than compositions of the prior art.Also as presented herein, it has been unexpectedly found that when thecompositions of the instant invention are formulated as a liquid ratherthan as a cream or lotion, a surprising and significantly more effectiveadministration of gNO is achieved, including a longer duration of gNOrelease and therefore the ability to use of a reduced amount or dosageof the composition. Moreover, unlike topical applications which areapplied directly to the lesion and therefore have an area of treatmentlimited to only the site of application, gNO released from a liquid NORScan also treat lesions or microbes that are not at the site ofapplication. For example, the liquid NORS can be sprayed into thenostrils of the subject, resulting in the extended release of gNO intothe subject's inspired air stream over minutes to hours. Furthermore,the duration of treatment can be reduced to a single treatment versusmultiple treatments over weeks to months, such as when a topicalapplication is used.

In one aspect, the present invention provides methods and compositionsuseful for the treatment of diseases and disorders where nitric oxidedelivery is beneficial. In one embodiment, the methods and compositionsof the present invention are useful for the treatment of a wound in asubject in need thereof. In one embodiment, the method further comprisescovering the wound with a gas impermeable cover. In another embodiment,the methods and composition are useful for treating fungal or diabeticassociated infections of the feet. It should be appreciated that theNORS of the present invention may be suitable for treating any infectioncaused by a microorganism or pathogen, including a bacterium, a virus, afungus, a protozoan, a parasite, an arthropod, and the like.

NO Releasing Solutions

The NORSs of the present invention provide an extended release of gNO toa subject in need thereof. By “extended release,” it is meant that aneffective amount of NO gas is released from the formulation at acontrolled rate such that therapeutically beneficial levels (but belowtoxic levels) of the component are maintained over an extended period oftime ranging from, e.g., about 5 seconds to about 24 hours, thus,providing, for example, a 30 to 60 minute, or several hour, dosage form.In one embodiment, the NO gas is released over a period of at least 30minutes. In another embodiment, the NO gas is released over a period ofat least 8 hours. In another embodiment, the NO gas is released over aperiod of at least 12 hours. In another embodiment, the NO gas isreleased over a period of at least 24 hours. An extended release NORS isbeneficial in that the solution can be administered to the subject overa short period of time, while the release of NO from the solutioncontinues following administration. Moreover, the use of an extendedrelease NORS allows the subject to remain ambulatory followingadministration of the solution, as opposed to remaining stationary whilebeing connected to a NO-releasing device in order to receive treatment.

In one aspect, the NORSs of the present invention have antibacterial,antifungal, and/or antiviral properties, and therefore may be useful asantibacterial, antifungal, and/or antiviral agents. In one embodiment,the NORS is an antibacterial agent effective against Acinetobacterbaumanii. In another embodiment, the NORS is an antibacterial agenteffective against Methicillin-resistant Staphylococcus aureus. Inanother embodiment, the NORS is an antibacterial effective againstEscherichia coli. In one embodiment, the NORS is an antifungal agenteffective against Trichophyton rubrum. In another embodiment, the NORSis an antiviral agent effective against Influenza H1N1.

The solution of the present invention becomes active when the nitritesand acids mix in saline or water in which the pH of the solution isbelow 4.0 and exhibits an increased or enhanced production level ofnitric oxide gas over an extended period of time. In one embodiment, thepH of the active state of the nitric oxide releasing solution is betweena pH of about 1.0 and a pH of about 4.0. In another embodiment, the pHof the active state of the nitric oxide releasing solution is between apH of about 3.0 and a pH of about 4.0. In one embodiment, the pH isabout 3.2. In another embodiment, the pH is about 3.6. In anotherembodiment, the pH is about 3.7. In one embodiment, the pH is about 4.0.In another embodiment, the pH is below about 4.0. Because the nitricoxide releasing solution of the present invention is not active untilthe acid interacts with the nitrites in liquid, the nitrite solution canbe pre-made, transported and set up for administration while in itsdormant state (pH greater than 4.0), without producing any appreciablenitric oxide gas or without losing its ability to produce an effectiveamount of nitric oxide gas. Then, when a user is ready to deliver oradminister the solution for treatment of a human subject, the solutioncan be activated immediately prior to administration to the humansubject by the addition of an acid (pH driven below 4.0), therebymaximizing the amount of nitric oxide gas produced by the administereddosage of solution.

In one embodiment, the pH of the solution can be lowered via addition ofat least one acidifying agent into the solution. Introduction of theacidifying agent drives the solution reaction towards the reactants,thus reducing the pH (creating more acid), which in turn creates morenitric oxide gas.

For example, by introducing sodium nitrite (or other salts of nitrites)to a saline solution it will very slowly produce nitric oxide gas, butin an undetectable amount (as measured by chemiluminescence analysismethodology (ppb sensitivity)). The rate of NO produced from thesolution increases as the pH is decreased, particularly as it dropsbelow pH 4.0. NO is produced based on the following equilibriumequations:NO₂ ⁻+H⁺→HNO₂  12HNO₂→N₂O₃+H₂O→H₂O+NO+NO₂  2a3HNO₂

2NO+NO₃ ⁻+H₂O+H⁺  2b

Therefore, an acidifying agent, for example an acid, may donate the H⁺to the nitrite (NO₂ ⁻). The more H⁺ present, the faster the reactionwill go towards HNO₂ and the more NO will be produced.

In one embodiment, the nitric oxide releasing solution includes the useof a water- or saline-based solution and at least one nitric oxidereleasing compound, such as nitrite or a salt thereof. In oneembodiment, the solution is a saline-based solution. In one embodiment,the nitric oxide releasing compound is a nitrite, a salt thereof, andany combinations thereof. Non-limiting examples of nitrites includesalts of nitrite such as sodium nitrite, potassium nitrite, bariumnitrite, and calcium nitrite, mixed salts of nitrite such as nitriteorotate, and nitrite esters such as amyl nitrite. In one embodiment, thenitric oxide releasing compound is selected from the group consisting ofsodium nitrite and potassium nitrite, and any combinations thereof. Inanother embodiment, the nitric oxide releasing compound is sodiumnitrite. In one embodiment, the solution is comprised of sodium nitritein a saline solution. In another embodiment, the solution is comprisedof potassium nitrite in a saline solution.

In one embodiment, the concentration of nitrites in the solution isbetween 0.07% w/v and about 0.5% w/v. In one embodiment, theconcentration of nitrites in the solution is no greater than about 0.5%w/v. In another embodiment, the concentration of nitrites in thesolution is about 0.41% w/v. In another embodiment, the concentration ofnitrites in the solution is between about 0.07-0.5% w/v. As used herein,the term “w/v” refers to the (weight of solute/volume of solution)×100%.

The solution of the present invention may also contain at least oneacidifying agent. As described elsewhere here, the addition of at leastone acidifying agent to the solution of the present inventioncontributes toward increased production of NO. Any acidifying agentwhich provides increased production of NO is contemplated by the presentinvention. In one embodiment, the acidifying agent is an acid.Non-limiting examples of acids include ascorbic acid, ascorbylpalmitate, salicylic acid, malic acid, lactic acid, citric acid, formicacid, benzoic acid, tartaric acid, hydrochloric acid, sulfuric acid, andphosphoric acid. In one embodiment, the acid is selected from the groupconsisting of ascorbic acid, citric acid, malic acid, hydrochloric acid,and sulfuric acid, and any combinations thereof. In one embodiment, theacid is citric acid.

As described above, the amount of acidifying agent present in thesolution will directly affect the rate of the reaction to produce NO. Inone embodiment, the amount of acidifying agent is no greater than about0.5% w/v. In another embodiment, the amount of acidifying agent is about0.5% w/v. In another embodiment, the amount of acidifying agent is about0.2% w/v. In one embodiment, the amount of acidifying agent is about0.07% w/v. In another embodiment, the amount of acidifying agent isbetween about 0.07-0.5% w/v.

The solution may be administered to the subject as an extended releaseformulation of NO gas, and optionally with a carrier formulation, suchas microspheres, microcapsules, liposomes, etc., as a topical ointmentor solution, or in an intranasal injection, as known to one skilled inthe art to treat a microbial disease or disorder.

The solution of the present invention may release a therapeuticallyeffective concentration of NO. In one embodiment, the therapeuticallyeffective concentration of NO is between about 100 ppm and about 1000ppm. In another embodiment, the therapeutically effective concentrationof NO is between about 120 ppm and about 400 ppm. In a preferredembodiment, the therapeutically effective concentration of NO is about160 ppm.

Methods

The present invention provides a method of treating a subject in needcomprising the delivery of a nitric oxide releasing solution to atreatment site of the subject. The present method can be used to treat,prevent, or reduce the incidence of any disease, disorder, or conditionwhere nitric oxide delivery is beneficial. Exemplary diseases,disorders, or conditions, include but are not limited to, respiratorydiseases, respiratory infections, wounds, burns, topical infections,inflammatory diseases, and the like. In a preferred embodiment, thedisease, disorder or condition is foot fungus. In another preferredembodiment, the disease, disorder or condition is diabetic foot ulcers.In another preferred embodiment, the disease, disorder or condition isinfected surgical wounds.

The present invention is unique in that it allows for delivery of nitricoxide to an ambulatory subject, or to an assembly line of subjects wherethe administration protocol for delivery of the NORS is accomplished ina short time period. This is particularly important and valuable whentreating humans, in that a patient need be only momentarily situated forthe short period of administration, and then can move about or be moved,as desired. For example, the extended release and delivery of nitricoxide to the treatment site by way of the administered nitric oxidereleasing solution allows for the treated subject to remain ambulatoryduring treatment, or stationary for a very short period of time. Thus,the subject is not constrained to a nitric oxide delivery device duringthe entire duration of nitric oxide delivery. Rather, the NORS can beadministered to the subject over a short duration of treatment, andfollowing administration the NORS will continue to deliver an extendedrelease of a therapeutically effective amount of nitric oxide to thesubject. In a preferred embodiment, the subject is a human.

In one aspect, the present invention includes a method for the treatmentof a wound in a subject in need thereof. In one embodiment, the methodof the present invention comprises spraying the wound of a subject witha nitric oxide releasing solution that has been prepared just prior toapplication and then covered with a gas impermeable or semi-impermeablycover that will retain the produced nitric oxide under the cover andtherefore expose the wound to the therapeutic concentration of nitricoxide for an extended period of time. The cover may have a small bleedhole to control or limit the pressure under the cover. This allows thesubject to be treated and then be ambulatory, eliminating the need forthe subject to remain next to the gas source.

In one embodiment, the method comprises the treatment of a wound,including but not limited to, an open wound, cut, scrape, burn, abscess,lesion, surgical wound, trauma wound, disease-associated wound or thelike. In certain embodiments, the method comprises administering thedormant solution to the treatment site. In certain embodiments, theacidifying agent is added to the dormant solution which lowers the pH ofthe dormant solution thereby creating the nitric oxide releasingsolution. For example, in one embodiment, the nitric oxide releasingsolution is produced by adding the acidifying agent to the dormantsolution directly on the treatment site. In another embodiment, thenitric oxide solution is produced away from the treatment site, and isthen topically applied to the treatment site. In one embodiment, themethod comprises administering a gas impermeable cover over thetreatment area of the subject, in order to constrain the produced nitricoxide gas over the treatment site. The cover may be applied prior to,during, or after administration of the dormant solution or nitric oxidereleasing solution. The nitric oxide releasing solution provides forextended nitric oxide production, thereby providing continuous deliveryof therapeutic nitric oxide to the wound of the subject.

Patients with open wounds resulting from physical injury or infection orfrom the result of known diseases such as diabetes or venous stasisdisease, have the need to have their wounds treated with a nitric oxidegas or nitric oxide compound. Because the NORSs of the present inventionprovide an extended release of nitric oxide, and thus require a shortduration of time for administration of the solution, subjects treatedwith an NORS of the present invention can remain ambulatory followingadministration of the solution. Therefore, the present invention isadvantageous over prior methods, where patients being treated withnitric oxide gas are required to remain stationary in a location wherethe delivery device and high pressure gas source are connected to theirwound.

In one embodiment, the present invention provides a method of treatingskin inflammation, including inflammation associated with psoriasis,dermatitis (atopic, contact, sebborheic, etc), eczema, tinea pedis, androsacea. In certain embodiments, the method comprises administering thedormant solution to the treatment site. In certain embodiments, theacidifying agent is delivered to the dormant solution which lowers thepH of the dormant solution thereby creating the nitric oxide releasingsolution. For example, in one embodiment, the nitric oxide releasingsolution is produced by applying an acidifying agent to the dormantsolution directly on the treatment site. In another embodiment, thenitric oxide solution is produced away from the treatment site, and isthen topically applied to the treatment site. In one embodiment, themethod comprises administering a gas impermeable cover over thetreatment area of the subject, in order to constrain the produced nitricoxide gas over the treatment site. The cover may be applied prior to,during, or after administration of the dormant solution or nitric oxidereleasing solution. The nitric oxide releasing solution provides forextended nitric oxide production, thereby providing continuous deliveryof therapeutic nitric oxide to the treatment site of the subject.

In certain embodiments, the nitric oxide releasing solution is preparedjust prior to administration to the subject through the administrationof an acidifying agent to a dormant solution. For example, as describedelsewhere herein, administration of the acidifying agent to the dormantsolution results in the lowering of the pH of the dormant solution,thereby activating the nitric oxide releasing solution to beadministered to the treatment site. Importantly, the nitric oxidereleasing solution provides for extended production of nitric oxide. Inone embodiment, the nitric oxide releasing solution produces nitricoxide for a period of between 1 minute and 24 hours. In one embodiment,the nitric oxide releasing solution produces nitric oxide for a periodof between 10 and 45 minutes. In one embodiment, the nitric oxidereleasing solution produces nitric oxide for at least 15 minutes. In oneembodiment, the nitric oxide releasing solution produces nitric oxidefor at least 30 minutes. In another embodiment, the nitric oxidereleasing solution produces nitric oxide for at least 1 hour. In anotherembodiment, the nitric oxide releasing solution produces nitric oxidefor at least 4 hours. In another embodiment, the nitric oxide releasingsolution produces nitric oxide for at least 8 hours. In anotherembodiment, the nitric oxide releasing solution produces nitric oxidefor at least 12 hours. In another embodiment, the nitric oxide releasingsolution produces nitric oxide for at least 24 hours. Thus, theadministered nitric oxide releasing solution provides for continuousdelivery of nitric oxide to the treatment site of the subject.

The nitric oxide releasing solution may be administered to the subjectin a variety of forms. The nitric oxide releasing solution may beadministered as a liquid, a spray, a vapor, micro-droplets, mist,footbath or any form which provides the release of nitric oxide from thesolution, as would be understood by one skilled in the art. In oneembodiment, the nitric oxide releasing solution is administered as aspray. In another embodiment, the nitric oxide releasing solution isadministered as a vapor. The amount or dosing volume of administerednitric oxide releasing solution may be varied in order to optimize theduration of nitric oxide production and delivery. In one embodiment, theamount of nitric oxide releasing solution administered to a subject isbetween about 0.1 mL and 5000 mL. In another embodiment, the amount ofnitric oxide releasing solution administered to a subject is betweenabout 10 mL and 1000 mL. In one embodiment, the amount of nitric oxidereleasing solution administered to a subject is about 2 mL. In oneembodiment, the amount of nitric oxide releasing solution administeredto a subject is about 10 mL. In one embodiment, the amount of nitricoxide releasing solution administered to a subject is about 32 mL. Inanother embodiment, the amount of nitric oxide releasing solutionadministered to a subject is about 160 mL. The nitric oxide releasingsolution may be readministered one or more times, as necessary toeffectively treat the subject. In one embodiment, the nitric oxidereleasing solution is administered once to a subject. In anotherembodiment, the nitric oxide releasing solution is administered multipletimes to a subject, where the NORS is readministered substantially aftercompletion of the extended release of gNO from the prior dosageadministered.

In certain embodiments, nitric oxide releasing solution is directlyadministered into the upper respiratory tract of the subject. Forexample, in one embodiment, the nitric oxide releasing solution issprayed into the upper respiratory tract of the subject. The solutionmay be administered into the upper respiratory tract of the subject oncean hour, once a day, once a week, once every two weeks, once a month,once every two months, once a year, and any and all ranges therebetweenas required to treat the subject. In one embodiment, the solution issprayed once a week. In another embodiment, the solution is sprayed oncea week for four consecutive weeks. The nitric oxide releasing solutionprovides for extended nitric oxide production, thereby providingcontinuous delivery of therapeutic nitric oxide to the upper respiratoryinfection of the subject.

The duration of administering the nitric oxide releasing solution to thesubject may be varied in order to optimize delivery. In one embodiment,the nitric oxide releasing solution is administered to the subject overa time period of less than 5 seconds. In another embodiment, the nitricoxide releasing solution is administered to the subject over a timeperiod of about 5 seconds. In another embodiment, the nitric oxidereleasing solution is administered to the subject over a time period ofabout 30 seconds. In another embodiment, the nitric oxide releasingsolution is administered to the subject over a time period of about 1minute. In another embodiment, the nitric oxide releasing solution isadministered to the subject over a time period of about 2 minutes. Inanother embodiment, the nitric oxide releasing solution is administeredto the subject over a time period of about 10 minutes. In anotherembodiment, the nitric oxide releasing solution is administered to thesubject over a time period of about 30 minutes.

In one embodiment, the method comprises the treatment, prevention, orreduction of incidence of a respiratory disease or disorder in asubject. Exemplary respiratory diseases or disorders treated by way ofthe present method include, but are not limited to emphysema, chronicbronchitis, asthma, adult respiratory syndrome (ARDS), chronicobstructive pulmonary disease (COPD), cystic fibrosis, influenza, andthe like. In certain embodiments, the method comprises the treatment ofa respiratory disease or disorder caused by a bacterial, fungal or viralinfection. In some embodiments, the infection is caused by a bacterium.In other embodiments, the infection is caused by a virus. Treatment of arespiratory disease by way of the present invention comprises thedelivery of a nitric oxide releasing solution into the upper respiratorytract of the subject to be treated. For example, in certain embodiments,the nitric oxide releasing solution may be injected, sprayed, inhaled,or instilled into the respiratory tract of the subject. The nitric oxidereleasing solution may be administered to the respiratory tract of thesubject via the nasal cavity or oral cavity of the subject. In oneembodiment, the nitric oxide releasing solution is sprayed into theupper respiratory tract of the subject. In one embodiment, the solutionis administered to the subject intranasally. In one embodiment, thesolution is administered to the sinuses. The nitric oxide releasingsolution provides for extended nitric oxide production, therebyproviding continuous delivery of therapeutic nitric oxide to the upperrespiratory tract of the subject.

In one embodiment, the method comprises the treatment of a wound,including but not limited to, an open wound, cut, scrape, burn, abscess,lesion, surgical wound, trauma wound, disease-associated wound whereinthe wound is caused by or affected by an infection. For example, theinfection may be caused by a fungus or a bacterium, including abacterium that has developed resistance to one or more antibiotics. Inone embodiment, the bacterium is S. aureus.

In one embodiment, the method comprises the treatment, prevention, orreduction of incidence of a respiratory disease or disorder in asubject, wherein the disease or disorder is caused by an infection. Forexample, the infection may be caused by a virus, a fungus, a protozoan,a parasite, an arthropod or a bacterium, including a bacterium that hasdeveloped resistance to one or more antibiotics. In some embodiments,the infection is caused by a bacterium. In other embodiments, theinfection is caused by a virus.

In one embodiment, the method comprises the treatment, prevention, orreduction of incidence of an infection in a subject, includinginfections caused by a virus, a fungus, a protozoan, a parasite, anarthropod or a bacterium, including a bacterium that has developedresistance to one or more antibiotics. In some embodiments, theinfection is caused by a bacterium. In one embodiment, the bacterium isAcetobacter baumanii. In another embodiment, the bacterium isMethicillin-resistant Staphylococcus aureus. In another embodiment, thebacterium is Escherichia coli. In other embodiments, the infection iscaused by a virus. In one embodiment, the virus is Influenza H1N1. Inother embodiments, the infection is caused by a fungus. In oneembodiment, the fungus is Trichophyton Rubrum.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein. Unless otherwise specified,the NORS as described in the following experiments is a saline basedsolution having a citric acid concentration of about 0.2% and sodiumnitrite concentration of about 0.41% (60 mM).

Example 1: Extended Release of NO from NORS

The materials and methods employed in these experiments are nowdescribed.

A NORS solution was prepared at a nitrite strength of 0.3% w/v and pH3.7. Once ready, a 3×3 in gauze was dipped into the solution, lightlysqueezed to discard excess liquid and placed in a “Hath Bath” device(FIG. 1). At different time points, the NO that was being released wasmeasured with a chemiluminescence analyzer (NOA 280i, General Electric,CO).

The results of the experiments are now described.

FIG. 2 shows the amount of NO detected at 3, 8, 15 min as well as 3, 4(2A) and 24 (2B) hours. The X scale is TIME (minutes) from start onmeasuring point (showing pre-measuring amount as 0-0.1 ppm) and Y scaleshowing amount of NO (measured in ppb).

The Chemiluminescent analyzer has a sample draws rate of 200 cc per minand thus, there is an initial peak and reduction in NO concentrationfollowing that. The “Hathback” may not be completely sealed and thussome NO may “escape”. However, release of NO was still detected 24 hoursafter gauze was saturated with the NORS solution.

Example 2: Antibacterial Efficacy of NORS on Acetobacter baumanii,Methicillin-Resistant Staphylococcus aureus and Escherichia coli

All of the following bacteria are common in wound infections:

A. baumannii is a species of pathogenic bacteria, referred to as anaerobic gram-negative bacterium, which is resistant to most antibiotics.Reported to cause infections among American soldiers wounded in Iraq.

E. coli—gram negative, common bacteria.

S. aureus is a common cause of surgical-site infection. It's a grampositive and it is frequently part of the skin flora.

Methicillin-Resistant Staphylococcus aureus—MRSA is, by definition, a S.aureus bacteria that has developed resistance to beta-lactam antibioticswhich include the penicillins (methicillin, dicloxacillin, nafcillin,oxacillin, etc.) and the cephalosporins.

The materials and methods employed in these experiments are nowdescribed.

Bacterial Preparation

A. baumanii, MRSA and E. coli bacterial culture were obtained fromAmerican Type Culture Collection (ATCC #BAA-747, #700698 and #25922).Bacteria were grown in Lysogeny broth (LB) (E. coli and A. baumanii) orBrain-Heart Infusion Broth (BHI) (MRSA) to 0.5 McFarland standard. 1 mLaliquots of these preparation containing approximately 2.5×10⁸ cfu/mLwere stored at −70° C. On the day of the experiments the fresh stock wasremoved from the freezer, thawed, and 2 mL of LB or BHI was added.Cultures were further diluted with LB or BHI to 10⁶ colony forming unitsper milliliter (cfu/mL).

NORS Preparation and Testing Procedure

NORS was prepared by mixing a specific concentration of sodium nitrite(0.07-0.41%) in saline and then reducing the pH to 3.7 with citric acid.Controls were—saline, sodium nitrite at 0.41% and pH of 6, and saline atpH 3.7 (reduced with citric acid).

100 μl of bacteria (10⁶ cfu/mL) was mixed with 900 μl NORS. After 10min, samples were serially diluted and plated on either LB or BHI agarplates. Cultures were incubated at 37° C. overnight (O/N) and then cfuscounted to quantify bacterial growth. Each experiment was done intriplicate and each experiment repeated three times.

The results of the experiments are now described.

NORSs comprised of citric acid and each with a different concentrationof nitrites in saline solution were tested on 3 different bacteriaspecies (A. baumanii, MRSA and E. coli) at 10 min exposure time in orderto evaluate antibacterial efficacy of the NORS. 0.41% nitrites at pH 3.7(0.2% w/v citric acid) resulted in complete eradication of all threebacteria (FIGS. 3 and 4).

Example 3: Antibacterial Efficacy of NORS on Mannheimia Haemolytica

The main bacterial pathogen of BRDc is M. haemolytica, which produces apotent leukotoxin that is its principal virulence factor. In this studythe effect of NORS on bacteria that is associated with bovinerespiratory infections was tested to demonstrate the overalleffectiveness of the present invention against diseases also found inother mammalian species.

The materials and methods employed in these experiments are nowdescribed.

Bacterial Preparation

M. haemolytica bacterial cultures were isolated and obtained from theAgriculture and Agri-Food Canada Research Centre (Lethbridge, Canada).Bacteria were grown to 0.5 McFarland standard. 1 mL aliquots of thesepreparations containing approximately 2.5×10⁸ cfu/mL were stored at −80°C. On the day of the experiments the fresh stock was removed from thefreezer, thawed, and 2 mL of BHI was added. Cultures were furtherdiluted with BHI to achieve OD₆₀₀ of 0.1. Two different serotypes of M.haemolytica were used. These serotypes were originally isolated frombovine nasopharyngeal swabs, and subsequently confirmed by biochemicaland polymerase chain reaction (PCR) assays as M. haemolytica (Klima etal., 2011, Vet. Microbiol. 149:390-398). They were serotyped in thelaboratory, against reference sera, which was generated in rabbits.

Antibacterial Effect of NORS on M. Haemolytica

NORS at different strengths was tested for efficacy against M.haemolytica serotypes. Saline was used as control. NORS (900 μl) wasadded to separate 1.5 mL sterile Eppendorf tubes. One hundred pi ofculture containing each serotype at 10⁶ CFU/mL (OD₆₀₀ 0.1) was thenadded to each tube and incubated for 30 seconds, 1, 2, 5 and 10 minutes.Following incubation, samples from each tube were serially diluted andwere plated on both BHI and blood agar sheep plates. Plates wereincubated at 37° C. overnight (O/N). Each experiment was done intriplicate and each experiment repeated three times.

The results of the experiments are now described.

It was observed that using NORS, even for 0.5 min, resulted insignificant (P<0.05) inhibition of M. haemolytica, compare to thecontrol. Using NORS for 1 minute caused a complete eradication of oneserotype of this bacteria and 2 minutes for both serotypes (FIG. 5).Both serotypes that were used here are isolates from feedlot cattle.

Example 4: Antiviral Efficacy of NORS on H1N1

For centuries influenza has affected human health both seasonally andwith recurring pandemics. Despite significant reduction of diseaseburden through vaccination efforts, circulation of seasonal influenza Avirus cause excess morbidity and mortality, particularly in patientswith preexisting pulmonary conditions.

The materials and methods employed in these experiments are nowdescribed.

Cell Lines & Viruses

Madin-Darby Canine Kidney Epithelial (MDCK) cells (ATCC CCL-34) wereobtained from the American Type Culture Collection and maintained inDulbecco minimal essential medium (DMEM) supplemented with 5% fetalbovine serum (FBS) and incubated at 37° C. in a humidified atmospherewith 5% CO₂ without antibiotics or antimycotic agents. MDCK cells weregrown as monolayers in 75-cm² cell culture flasks. Passages between 3and 15 were used for these experiments.

Viral strain was obtained from the laboratory stock from the BritishColumbia Center for Disease Control. Stocks of influenza A viruses,A/Denver/1/1957 (H1N1), were grown in MDCK for 48 hours, with mediumcontaining 2 μg/mL modified trypsin (treated with TPCK) without serum.Stock virus was prepared as clarified cell-free supernatants. Virusconcentration for stocks was determined by standard plaque assay on MDCKcells [27]. Virus titer for this stock was 6×10⁶ plaque forming units(PFU)/mL respectively.

Experimental Protocol

Aliquots of virus, diluted in phosphate buffer solution (PBS), usually20 μL, were spotted onto the appropriate sterile glass surface, spreadinto a film by means of a sterile tip, and allowed to dry, within abiosafety cabinet (normally 15-20 min). Each sample received 2 sprays(100 μL) of different concentration of NORS (0.007-0.14% w/v) at pH 3.7.Controls consisted of equivalent samples sprayed with just saline,nitrites (0.14% at pH 6) and saline at pH 3.7. After 5 min, all samples,and equivalent control samples were reconstituted in 1.0 mL PBS andassayed by plaque formation (plaque forming units, pfu) in theappropriate cells.

The results of the experiments are now described.

NORSs comprised of citric acid and each with a different concentrationof nitrites in saline solution were tested on Influenza H1N1 in order toevaluate the antiviral efficacy of the NORS. A strength of 0.07% w/vnitrites at pH 3.7 (0.08% w/v citric acid) resulted in over 90%reduction (FIGS. 6 and 7A), while 0.14% caused complete eradication ofthe virus (FIGS. 6 and 7B).

Example 5: Antiviral Effect of NORS on Infectious BovineRhinotracheitis, Bovine Respiratory Syncytial Virus and BovineParainfluenza-3

It is clear that in cattle, as in humans and other mammalian species, anactive viral infection dramatically increases susceptibility tocontracting bacterial pneumonia (Bedling and Slifka, 2004). This hasbeen demonstrated experimentally in cattle infected with any one ofseveral bovine respiratory viruses such as bovine herpes virus 1 (BHV-1)or bovine respiratory syncytial virus (BRSV), after which renders cattlehighly susceptible to a secondary bacterial infection when challengedwith M. haemolytica (Hodgson et al., 2005; Yates 1982). Theseobservations suggest that viral infection impairs host defensemechanisms against M. haemolytica, or amplifies undesirable aspects ofthe host response to this bacterial pathogen. In this example the effectof NORS on 3 viruses related to bovine respiratory infections wastested.

The materials and methods employed in these experiments are nowdescribed.

Cells and Viruses

Madin-Darby bovine kidney (MDBK) cells (ATCC CCL 22) were grown inEagle's minimum essential medium (MEM) containing 10% fetal bovineserum. Infectious Bovine Rhinotracheitis (IBR), Bovine RespiratorySyncytial Virus (BRSV) and Bovine parainfluenza-3 (PI-3) were usedthroughout the experiments. These viruses were propagated in MDBK cellsin Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% fetalbovine serum and stored at −80° C. until use. The amount of virus wasmeasured by a plaque assay on MDBK cells.

Direct Virucidal Activity of NORS

Virucidal activity was tested using equal volumes (0.025 mL) of virussuspension, containing 10³ to 10⁷ plaque-forming units (PFU/mL) of eachof the 3 viruses and NORS. The two volumes were mixed together andincubated for 1 or 10 minutes at room temperature. The viruses werediluted with PBS containing 2% fetal bovine serum (FBS) and the numberof infectious virus in each preparation was measured by a plaque assay.

The results of the experiments are now described.

IBR is the most susceptible virus to NORS, with a complete eradicationwith all initial titers after 10 min exposure and a significant (P<0.05)reduction for all titers after 1 min (FIG. 8A). PI3 was a bit lesssusceptible but significant reduction in viability at all titers wasobserved, both after 1 and 10 min (FIG. 8C). The least susceptible viruswas BRSV, where no significant difference was observed after 1 minexposure, although a significant (P<0.05) reduction in viability wasobserved following 10 min exposure at all titers (FIG. 8B). The abilityof NORS to eradicate the virus was found to be in direct correlationwith the initial titer.

Example 6: Antifungal Efficacy of NORS

Most cutaneous infections are the work of the homogeneous group ofkeratinophilic fungi known as dermatophytes. Tinea pedis, known asAthlete's Foot, is the most prevalent form of superficial mycoticinfections of the (Drake et al., 1996). Species from the genusTrichophyton are most commonly isolated from clinical samples, withTrichophyton rubrum and Trichophyton mentagrophytes being most common(Drake et al., 1996; Baran and Kaukhov, 2005).

Published paper-Regev-Shoshani et al., 2013, J. Appl. Microbio.114:536-544

The materials and methods employed in these experiments are nowdescribed.

Fungal Preparation.

Trichophyton rubrum (18758) and Trichophyton mentagrophytes (114841)were obtained from the American Type Culture Collection (ATCC). Fungiwere grown at 30° C. in Sabouraud Broth (SAB) for three days to amycelial biomass of 1 mg/mL. Experiments on mycelial viability were donewith this concentration. Conidia were isolated by shaking (on a Fishershaker at 100 RPM) glass beads (Soda Lime 2 mm, VWR) for 60 seconds onthe surface of mycelia grown on SAB agar plates for a minimum of sevendays. Conidia covered glass beads were vortexed in sterile saline tosuspend conidia in solution.

Preparation of NORS.

Nitric oxide releasing solutions (NORS) were prepared utilizing sodiumnitrite and citric acid, as previously described. Specifically, this wasdone by dissolving solid sodium nitrite (NaNO₂) into sterile distilledwater (dH₂O) to reach a final concentration of 0.007-0.14% w/v. Then,those solutions were acidified to pH 3.7 using a predetermined mass ofcitric acid (up to 0.1%).

NORS Antifungal Effect on Mycelial Viability of T. rubrum and T.mentagrophytes.

NORS containing NaNO₂ at concentrations of 0.007, 0.14, 0.35, 0.7% w/vwere tested for their efficacy as antifungal agents. Sterile water (pH6) was used as control. Sterile water adjusted to pH 3.7 using citricacid, and sterile water with 0.14% NaNO₂ (pH 6) were tested as well todetermine whether either solution possessed a fungicidal effect bythemselves. NORS (4 mL) was prepared and added to separate 5 mL sterileplastic tubes. One hundred μl of culture containing mycelia at a biomassof 1 mg/mL was then added to each tube and incubated for 10, 20 and 30minutes. Following incubation, samples from each tube were seriallydiluted and were plated on SAB agar plates. Plates were incubated at 30°C. until growth could be detected and counted (about 3 days for T.rubrum and 2 days for T. mentagrophytes). Each experiment was done intriplicate and repeated three times.

A set of control experiments were done in order to eliminate thepotential antifungal effect of the citric acid concentration in thetreatment solution. Different concentrations of citric acid wereprepared and pH was raised to 3.7 using NaOH. The same experimentalmethodologies with water as a control were used to perform these tests.

Gaseous Oxides of Nitrogen Produced from NORS and its Effect on MycelialGrowth.

The concentration of NO and other gases released from the NORS into thehead space were determined by gas chromatography with a massspectrometer detector (GC-MS). NORS (0.14% nitrites w/v) was preparedinside the sterile 5 mL plastic tubes described above. Each tube wasthen sealed for 30 minutes after which, 1 mL of the head space above thesolution was analyzed by GC-MS. GC-MS (Varian™ CP-3800 Gas Chromatographconnected to a Varian™ 1200 Quadrupole MS) analysis was performed usinga standard method that had previously been created and calibrated toseparate and quantify NO, NO₂ and N₂O molecules, using calibrationgases. The method was set to a constant temperature of 31° C. with asampling flow rate of 1 mL/min with helium gas as the carrier gas.Injector temperature was set to 120° C.

In order to demonstrate that NO, found in the headspace, is responsiblefor the fungicidal effect of NORS, mycelia of T. mentagrophytes (10 mLat 1 mg/mL) were combined with 20 mL of sterile saline inside a sterileglass test tube connected via Teflon tubing to a separate glassapparatus, as illustrated in FIG. 9. Sterile saline (0.9% sodiumchloride) was used in replacement of sterile dH₂O in order to ensure anyfungicidal activity measured was not the result of osmotic imbalances.NORS was added to the glass apparatus using a 50 mL syringe through the‘fill port’ (FIG. 9) then sealed using paraffin laboratory film andplastic wrap. A higher strength NORS was required to produce asufficient volume of gas to account for the much greater head spacevolume in the apparatus as opposed to the 5 mL tubes previouslydescribed. The apparatus was then left at room temperature for 2, 4, 8,16 and 24 hours (each performed separately) after which, samples fromthe glass test tube were plated onto SAB agar plates, incubated at 30°C. for 48 hours and fungal viability determined. The growth from theexposed test tube was compared to a control of the same contents keptalongside the exposure in a sealed glass test tube (FIG. 9). Anothercontrol study was performed with the same apparatus, using salineinstead of NORS. Nitrite concentration in the attached glass test tubewas measured after each time point, using Griess reagent (Green et al.1982).

The results of the experiments are now described.

NORS Antifungal Effect on Mycelial Viability of T. rubrum and T.mentagrophytes.

T. rubrum and T. mentagrophytes were grown from conidia for a minimum of72 hours to a mycelial biomass of 1 mg/mL. Mycelia was added totreatment or control tubes and incubated for up to 30 minutes, afterwhich, samples were plated and concentration (cfu/mL) was determined. AsNORS is formulated from nitrites and citric acid (lowering pH to 3.7),the individual exposure effect of water at pH 3.7 and 0.14% w/v nitritesat pH 6 was tested and compared to an appropriate control. Minimal to noeffect was detected after a 30 minute exposure with either 0.14% sodiumnitrite (pH 6) or citric acid at pH 3.7.

FIG. 10 shows the mycelia viability following exposure as a percentageof control. T. mentagrophytes (FIG. 10A) and T. rubrum (FIG. 10B) bothdemonstrated similar responses to different concentrations of NORS. Bothspecies were tolerant to up to 0.014% w/v nitrite at pH 3.7 for up to 20minutes demonstrating a reduction of less than 25%. While using a higherconcentration of 0.035% w/v nitrite at pH 3.7 rendered a time dependentfungicidal effect starting from a significant 25% reduction after 10minutes and reaching a 98% reduction after 30 minutes for both species.An increase to 0.7% w/v nitrite and 0.08% w/v citric acid was highlyeffective at eradicating mycelia resulting in a greater than 99%reduction at 10 minutes and complete kill at 30 minutes for T.mentagrophytes and a complete kill at all time points for the T. rubrum.Not surprisingly a concentration of 0.14% w/v nitrite at pH 3.7 showed acomplete kill, even after 10 minutes, for both organisms (not shown ongraph).

Controls with citric acid at pH 3.7, and nitrites alone, had nosignificant effect on mycelial growth when compared to water control.

Analysis of Head Space Gases Above NORS Using GC-MS.

A head-space sample from the tube (containing 4 mL of 0.14% NORS) after20 minutes was analyzed by GC-MS to determine which gaseous moleculescould be detected. Specific detection was set to identify NO, NO₂, N₂Oand CO₂ and their respective concentrations were determined. As revealedby the chromatogram in FIG. 11A, three types of gas molecules weredetected (excluding water vapor, not shown). NO eluted at 5.4 minutes,NO₂ at 5.98 and CO₂ eluted at 6.03 minutes. No other peaks were detectedin a scan program for MW 18-100. FIG. 11B shows the molecular weight of30 for the peak at 5.4 minutes, which correlates to NO. NO concentrationwas found to be 170(±30) ppm; NO₂ was 40(±10) ppm. CO₂ (coming fromambient air) was found as well but not quantified. N₂O was not detected.As a comparison, headspace from a control tube had only ambient levelsof CO₂ present in it.

In order to demonstrate that the NO being produced by the NORS is likelythe active agent responsible for the antifungal activity observed, anapparatus was constructed to ensure no direct contact occurred betweenfungal mycelia and the NORS, allowing only for the exchange of headspacegases (FIG. 9). The antifungal activity of the NORS headspace gases wastested on T. mentagrophytes mycelia at 1 mg/mL. Both mycelial viabilityand nitrite concentrations were measured after 2, 4, 8, 16, and 24hours. FIG. 12 illustrates the antifungal activity of the NORS gasesover a 24 hour period. Some antifungal effect was observed after 4 and 8hours of exposure, where a one log₁₀ reduction in mycelial viability wasobserved. Complete kill resulted after 16 hours of exposure. Myceliacontrols showed no significant change in concentration during these timeperiods. Nitrite concentrations were shown to inversely correlate withmycelial viability. Following 16 hours, where complete kill of themycelia was reached, a nitrite concentration of 0.014% w/v was measured.Mycelia controls showed nitrite concentrations to be negligible. Whenusing saline in the apparatus, instead of NORS, no mycelial kill wasfound and no NO was found in the headspace.

Example 7: Prophylactic Nitric Oxide Treatment Reduces Incidence ofBovine Respiratory Disease Complex in Beef Cattle Arriving at a Feedlot

In further demonstration of the overall effectiveness of the presentinvention to also combat similar diseases in mammalian species, theresults described herein demonstrate that NO treatment on arrival to thefeedlot significantly decreased the incidence of BRDc in beef cattle.Eighty-five, crossbred, multiple-sourced, commingled commercial weanedbeef calves were monitored and scored for temperature, white bloodcount, clinical score, hematology, cortisol levels andneutrophil/lymphocyte ratio. NO treatment or placebo was given once onarrival to the stockyard. After one week, 87.5% of sick animals werefrom the control while 12.5% were from treatment groups, and after twoweeks 72% and 28% respectively. Treatment was shown to be safe, causingneither distress nor adverse effects on the animals.

The materials and methods employed in these experiments are nowdescribed.

Animals and Management

Eighty-five, crossbred, multiple sourced, commingled commercial weanedbeef calves were obtain for these studies. All studies were conducted atthe Lacombe Research Centre beef research facility and all managementpractices followed Canadian Council of Animal Care guidelines (CanadianCouncil on Animal Care, 1993) and Canadian Beef Cattle Code of Practiceguidelines (Agriculture Canada, 1991). In addition, the researchprotocols were reviewed and approved by the Lacombe Research Centreanimal care committee. The calves were procured through a conventionalauction system and all animals had been exposed to between 4-6 h oftransport prior to the study. These calves were chosen in order toprovide study groups displaying a BRDc incidence range of 30-60% whichis typical of the beef industry in Canada for these “put together” herdsof cattle. On arrival at Lacombe the calves were off loaded, weighed,sampled for saliva and blood using procedures described previously(Schaefer et al., 2012, Virulence 3:271-279).

The calves were randomized into treatment and control groups, labeledwith color coded ear tags and numbers. Animals were then placed intooutdoor pens measuring approximately 60×60 meter and were bunk feed adlibitum a balanced cereal silage diet, which met or exceeded NationalResearch Council recommendations (NRC, 1984, Nutrient Requirements ofBeef Cattle, 6th ed. National Academy Press, Washington, D.C.). Theanimals also had free access to water and were provided a straw beddingarea with a roof covering.

Clinical Scores

While contained in their receiving pens the calves were monitored dailyby trained personnel, whom were blinded as to the treatmentinterventions, for clinical signs of illness using methods describedpreviously (Schaefer et al., 2007, Res. Vet. Sci. 83:376-384). Briefly,clinical scores were designed to identify BRDc and were based on theappearance of four criteria as follows:

Respiratory insult: (0-5): 0=no insult, normal breath sounds (NBS);1=Very Fine Crackle (rale) (VFCR) on auscultation and/or a moderatecough; 2=Fine Crackle (subcrepitant) (FCR) on auscultation and/or amoderate nasal discharge and moderate cough; 3=Medium Crackle(crepitant) (MCR) on auscultation and/or a moderate to severe viscousnasal discharge with cough; 4=Course Crackles (CCR), tachypnea (>15% ofthe norm) and/or a severe nasal discharge with respiratory distress andobtunded lung sounds and 5=CCR with dyspnea, tachypnea, markedrespiratory distress and/or lung consolidation.

Digestive insult: (0-5): 0=no insult, normal, eating and drinking;1=mild or slight diarrhea with slight dehydration (<5%) and reducedeating; 2=moderate diarrhea with 10% dehydration and reduced feed intake(<50%); 3=moderate to severe diarrhea with 10% or less of feed intakeand more than 10% dehydration; 4=severe diarrhea, and less than 10% ofnormal feed intake and 5=severe diarrhea and not eating, not drinkingand dehydrated.

Temperature score: Core temperature (rectal) (0-5): 0=<37.7° C.;1=37.7-38.2° C.; 2=38.3-38.8° C.; 3=38.9-39.4° C.; 4=39.5-40.0° C. and5=>40° C. Rectal or core temperatures for the calves were collected atthe start and end of the study only as these were the times that theanimals were restrained. Disposition or lethargy score: (0-5): 0=nolethargy, normal posture; 1=mild anorexia or listlessness, depressedappearance; 2=moderate lethargy and depression, slow to rise, anorectic;3=recumbent or abnormal posture, largely depressed; 4=prostrate,recumbent or abnormal posture and 5=death.

Laboratory Analysis

With respect to laboratory analysis, salivary and serum cortisol levelswere analyzed using an enzymatic assay previously described (Cook etal., 1997, J. Ag. Food Chem. 45:395-399).

Hematology

Hematology values were measured on a Cell-Dyn 700 Hematology Analyzer(Sequoia—Turner Corp. Mountain View, Calif.). Differential blood cellcounts were determined utilizing stained blood smears (Geisma-Wrightquick stain) and direct microscope examination of 100 cells.

Clinical Rescue Treatment

Animals displaying overt clinical symptoms of BRDc as identified by ablinded pen keeper were rescued and subsequently received immediatetreatment as recommended by the Lacombe Research Centre veterinarianfollowed by continued monitoring and re-treatment if required. Theseanimals were classified as true positive (TP) in the statisticalanalysis.

Nitric Oxide Treatment

NORS was delivered with a spray device. The NORS is a saline basedsolution having a citric acid concentration of about 0.2% w/v and sodiumnitrite concentration of 0.41% w/v. This solution was previously testedand verified to release 160 ppm NO in a 3 L/m flow of medical air(Praxair, Canada), for 30 min. In brief, 32 mL of the solution wassprayed into a two inch diameter vinyl chloride tube and inserted intoenvironmentally controlled system (as previously described by Ghaffariet al., 2005) where NO was measured using chemiluminescence (SieversNitric Oxide Analyzer NOA 280i). Animals were restrained in aconventional hydraulic cattle-handling catch and given either a placebo(saline) or treatment (NO) by an individual blinded as to theintervention. Each animal received 1 spray (8 mL), alternating into eachnostril, twice, for a total of 32 mL before being released into thefeeding lot pen areas. The duration of treatment administration was lessthan 5 s.

Determination of True Positive (TP) and True Negative (TN) Animals forBovine Respiratory Disease Complex (BRDc)

The determination of an animal true positive or negative for BRDc wasbased on the comparison to a set of “gold standard” values using apreviously published method (Humblet et al., 2004, Res. Vet. Sci.77:41-47; Schaefer et al., 2007, Res. Vet. Sci. 83:376-384). Thisapproach is commonly promoted in both veterinary and human medicaldiagnostic studies (Galen and Gambino, 1975, Beyond Normality. J. Wileyand Sons, NY; Thrusfield, 1995, Diagnostic testing, p. 266-285,Veterinary Epidemiology, 2^(nd) ed. Blackwell Sci. Ltd., Oxford).

In the current study, the criteria for a true positive animal for BRDcwas defined as an animal displaying three or more of the followingsigns; a core temperature of >40° C. (or 103.5° F.), a white blood cellcount of less than 7 or greater than 11×1000/L, a clinical score of >3or a neutrophil/lymphocyte ratio of <0.1 (leucopenia) or >0.8(neutrophilia). A true negative animal was defined as an animaldisplaying a score of 0 or 1. These parameters were consideredconsistent with suggested normal and abnormal ranges (Kaneko, 1980,Clinical Biochemistry of Domestic Animals, Academic Press, NY; Blood etal., 1983, Veterinary Medicine, 6^(th) ed., Communications Branch,Agriculture Canada). For laboratory assessments, all calves weremonitored at the beginning of the study and again three to four weekslater.

The results of the experiments are now described.

Safety of NO Treatment

All animals tolerated the nitric oxide treatments well. Some of theanimals sneezed but none exhibited coughing or other clinical signs ofdistress. There were no adverse events nor serious adverse eventsobserved in either cohort. No animals died during the time of the study.Mean salivary and cortisol levels were equivalent in each group (Control5.4±5.7 nmol/L; Treatment 6.66±5.5 nmol/L) without a significantdifferences (p=0.09).

Decreased Incidence of BRDc

As can be seen in Table 1, during days 1-14, 13 animals from the controlgroup and 5 animals from the treatment group were identified as TP. Thetable shows values recorded for all 4 parameters determining TP/TN forall TP animals. Temperature, clinical score, white blood count,neutrophil/lymphocyte ratio were also included. All sick animals had 3or 4 parameters recorded below or above the defining value for TP. Thisscoring approach provides a more robust definition of sick animals ascompared to looking at just a temperature threshold alone. All animalshad clinical scores above 3 and 15 out the 18 animals had temperaturerecorded as 103.5° F. or higher. Thirteen out of the 18 TP animals werealso recognized by the pen keeper as sick.

TABLE 1 Parameters determining TP/TN Animal Day of Temp Clinical N/LPulled # sickness (° F.) Score WBC ratio out Control 1 14 104.2 8 9.580.027 Y 2 14 103.8 4 11.1 0.121 Y 3 14 103.6 5 5.97 0.139 Y 4 7 105.2 86.38 0.721 Y 5 2 104.1 8 7.97 0.092 Y 6 4 105.8 7 2.75 0.882 Y 7 10104.5 8 11.15 1.136 Y 8 9 103.5 7 6.61 0.099 Y 9 7 103.1 4 6.53 0.023 N10 7 103.1 4 10.95 0.037 N 11 7 103.5 5 7.56 0.077 N 12 7 103.4 5 5.990.092 N 13 8 105.6 9 4.67 0.678 Y Treatment 1 14 103.5 5 6.7 0.556 N 2 6106.2 8 12.15 1.058 Y 3 8 104.2 10 6.57 1.537 Y 4 11 105.4 9 11.25 0.877Y 5 8 104.6 8 6.72 0.191 Y

Values recorded for all 4 parameters determining TP/TN for all sickanimals in both groups during the first 2 weeks after arrival to feedlot(TP indicators are highlighted).

Table includes day of recorded sickness and whether the animal waspulled out by herdsman.

In terms of a BRDc incidence in this model, of these 82 calvesevaluated, after 7 days post arrival, 8 displayed true positive for BRDc(10%). As shown in FIG. 13A, 7 animal (17.5%) out of the 40 in thecontrol group and 1 (2.4%) out of 42 in the NO treated group wereidentified as TP in the first week. Of these 8 animals, one (12.5%) wasfrom the NO treated group and seven (87.5%) were from the saline controlgroup (FIG. 13B). These results demonstrate a very significant reductionof the incidence of BRDc between the treatment and control cohorts witha single NORS treatment upon arrival into the stockyard (p<0.001).During the first 14 days, 18 animals (22%) had an incidence of BRDc andof these 13 (72.2%) were in the control group whereas only 5 (27.8%)were in the treatment cohort (FIG. 13B).

These data, collected from three separate randomized and blinded studiesperformed in a conventional feedlot, show that NO significantlydecreased the incidence of BRDc, as defined by true positive rigor, by adifference of 75% as compared to a saline placebo (87.5% of sick animalswere from control group versus 12.5% from treatment group.

Example 8: Bioavailability of Nitric Oxide to Control Bovine RespiratoryDisease Complex in Calves Entering a Feedlot

The results described here demonstrate that the delivery of NORS to abovine's nostril is biologically available. Thirteen, crossbred,multiple-sourced, commingled commercial weaned beef calves were treatedmultiple times intranasally over a 4 week period with either a nitricoxide releasing solution (treatment) or saline (control). Exhaled NO,methemoglobin percent (MetHb) and serum nitrites demonstrated biologicalavailability as a result of treatment.

The materials and methods employed in these experiments are nowdescribed.

Animals and Management

The study was conducted at a commercially registered feedlot facility inWestern Canada (Westwold, British Columbia). All management practicesfollowed the Canadian Council of Animal Care guidelines (CanadianCouncil on Animal Care, 1993) and Canadian Beef Cattle Code of Practiceguidelines (Agriculture Canada, 1991). In addition, the researchprotocols adhered to the Experimental Study Certificate approved by theHealth Canada Veterinary Drug Directorate and the Thompson RiversUniversity animal care committee.

Thirteen, crossbred, multiple-sourced, commingled commercial weaned beefcalves were procured through a conventional auction system. All animalswere exposed to approximately 4-6 hours of transport prior to the study.These calves were chosen in order to provide study groups displaying aBRDc incidence range of 30-60% which is typical of the beef industry inCanada for this type of a cattle population. On arrival at the feedlotthe calves were off loaded, randomized into one of three cohorts,received ear tags, were vaccinated (Bovi-Shield® GOLD FP™ 5; Pfizer,INFORCE™ 3; Pfizer, Mannheimia Haemolitica Bacterin-Toxoid; Pfizer) andweighed.

Calves consisted of 3 groups as follows: 1) Control group—receivedsaline as placebo (n=4), 2) Treatment group—2 sprays of NO treatment ineach nostril—32 mL in total (n=5) and 3) Treatment group with 5 timesthe normal NO treatment dose of 160 mL in total in each treatment. Allgroups were treated with NO on arrival approximately 2 minutes aftergiving the vaccines. Animals were then placed into 2 outdoor corrals,separated into control or treatment groups. They were fed chopped hay,grain screening pellets, along with alfalfa/grass and barley silage toprovide a complete ration which met or exceeded National ResearchCouncil recommendations (NRC, 1984). The animals also had free access towater and were provided with sawdust bedding.

Nitric Oxide Treatment

A nitric oxide releasing solution (NORS) was prepared in a 5 L spraydevice, which contained 2 L of the NORS. The NORS is a saline basedsolution having a citric acid concentration of about 0.2% and sodiumnitrite concentration of about 0.41% (60 mM). The solution was preparedon site just prior to administration. This solution was previouslytested to release 160 ppm NO in a 3 L/min flow of gas as verified bychemiluminescence analysis (280i, General Electric, CO). Animals werebriefly restrained in a conventional hydraulic cattle handling squeezeand given either saline or NORS by a trained research assistant. Eachanimal in the control and normal treatment dosing groups received 1spray (8 mL), alternating into each nostril, for a total of 32 mL ofeither of the interventions before being released into the feeding lotpen areas. Each animal in the second dosing group received 5 times theabove-described dosing volume, for a total of 160 mL. Animals receivedthese treatments weekly for four consecutive weeks.

Laboratory Analysis

Blood samples were collected on day 14. Blood was collected by alicensed veterinarian via jugular venipuncture before treatment, 5minutes post treatment and 30 minutes post treatment interventions. Eachsample was placed in one of 3 appropriately prepared collectiontubes—one for each measurement: Cortisol, methemoglobin percent (MetHb)and nitrites. Serum cortisol was analyzed by Kamloops Large AnimalVeterinary Clinic LTD. (1465 Cariboo Place, Kamloops, BC V2C 5Z3). Allblood samples were transferred to Thompson River University (TRU) on icefor measurements of MetHb. Blood gas analysis was done for co-oximetricmeasurement of MetHb using an ABL 800 FLEX analyzer (Radiometer AmericaInc., Ohio, USA). Blood gases including arterial oxygen, carbon dioxide,pH, bicarbonate and electrolytes were also measured at that time.

Measurement of Exhaled NO

Fractional exhaled concentration of NO (F_(E)NO) was measured using achemiluminescence analyzer (280i, GE, CO). A F_(E)NO baselinemeasurement was obtained for each subject by recording for 1 minutebefore and after treatment intervention until F_(E)NO levels returnedback to baseline. The sampling tube had a water filter to prevent liquidfrom getting into the device. The filter was at the distal end and washeld as close as possible to the animal's nostril. All of the animalhandling was performed by the same person to reduce handler variation.The machine was calibrated before each use with standard calibrationgases as per manufacturer's instructions.

Nitrite Measurements

Blood samples for nitrite analysis were collected on day 14 (asdescribed above). All samples for nitrite measurement were placed inheparinized tubes and centrifuged for 5 minutes at 5000 RPM. Thesupernatant was recovered and placed in Eppendorff tubes, placed on dryice, and then samples were immediately transferred to a −80° C. freezeruntil processing. Nitrite measurements were performed using achemiluminescent liquid interface technique according to themanufacturer's instructions (280i, General Electric, CO).

The results of the experiments are now described.

Bioavailability Measurements

All three parameters measured for bioavailability (MetHb, F_(E)NO andnitrites in serum) showed biochemical changes within 5 minutes posttreatment.

MetHb Measurements

MetHb was measured using Cooximetry on blood samples taken beforetreatment and at 5 and 30 minutes post treatment with either NORS orsaline control. The saline control group (FIG. 14A) did not have anysignificant difference between MetHb values before and after treatment.On the other hand, the NORS treatment group had higher values of MetHb 5and 30 minutes after administering the treatment (FIG. 14B). FIG. 14depicts the values of MetHb for the control and treatment group animals(FIGS. 14A and 14B, respectively), and the average difference betweenthe MetHb value at 5 and 30 minutes post treatment, compared to baseline(FIG. 14C). There was a significant difference observed at the 5 minutepost-treatment time between the NORS and the saline control group. TheMetHb value was, on average, 4.8 points higher in the treatment groupcompared to 0.1 lower in the control group. Small but insignificantdifferences were found after 30 min between treatments, although for theNORS treatment group, values stayed significantly higher than thebaseline measure.

Measurement of F_(E)NO

When administering NORS to the animal, the F_(E)NO were high enough tobe detected by chemiluminescent analysis within seconds to minutesfollowing the NORS treatment (FIG. 15). FIG. 15A depicts the F_(E)NOmeasured after giving saline to the animal (2.4 ppb) while FIG. 15Bshows F_(E)NO after giving NORS (around 400 ppb for approximately 5minutes). This was measured outside the nostril, while diluted with air,and thus levels are much lower than actual F_(E)NO levels. However, thisresult demonstrates that the NO gas is present compared to the salinecontrol.

Nitrites in Serum

Nitrites were measured using the chemiluminescence liquid interfacetechnique. Samples were extracted with cold ethanol and 50 μl wasinjected. As seen in FIG. 16, 5 minute post treatment there was a raisein the nitrite concentration in the animal's serum. By 30 minutes posttreatment there was no significant difference (P>0.05) from the controlgroup.

These results described herein demonstrate that NORS at 0.41% resultedin NO bioavailability, which was confirmed by the rise of F_(E)NO in thetreated animal and by the expected transient rise in MetHb percent,indicating that NO was available within the respiratory tract andmetabolized in the serum into increased nitrite levels.

Example 9: Ferrets Study with NORS

This experiment was performed to test the effect of NORS on temperatureand blood nitrites in ferrets, after Influenza A infection.

The materials and methods employed in these experiments are nowdescribed.

Six 10-12 week-old ferrets were purchased from Triple F Farms andacclimated for 5-7 days prior to challenge. They were housed loose andtogether in a 12×18 ft. room for the duration of the study.

Six ferrets were stable for the study were anesthetized withketamine-xylazine and bled for nitrite baseline values. All animals werechallenged with Influenza A/California/04/2009 virus by intranasalinstillation of approximately 7.5×10⁴ pfu in 0.5 mL. Animals were thenbled again to assess serum nitrite levels. Blood samples were obtained30 and 240 minutes post treatments and analyzed for serum nitrites witha chemiluminescent analyzer. Within 5-10 minutes of inoculationtreatment interventions were administered. One cohort of ferrets (n=3)received approximately 2 mL NORS over a ten minute period with a smallvolume nebulizer at 7 L/min. Another cohort of ferrets (n=3) receivedapproximately 2 mL saline over a ten minute period with a small volumenebulizer at 7 L/min (FIG. 17)

The results were analyzed using the unpaired Student's t-test forcomparison between any two groups. Group means were statistically testedby least squares means (two-tailed t-test). For experiments withmultiple (more than 2) sets, Statistical analysis of data obtained wereperformed using a one-way analysis of variance (ANOVA) and Tukey'sMultiple Comparison Test Data analysis and graphical presentation weredone using a commercial statistics package (Graphpad-Prism V 3.0,GraphPad Software Inc., USA). Unless otherwise specified, p<0.05indicated statistical significance. Results were reported as themean±standard deviation.

The results of the experiments are now described.

Nitrites in the serum were significantly elevated at 30 minutes (p<0.05)and returned to baseline after 240 minutes post NORS treatment ascompared to baseline values (FIG. 18A). Moreover, when the NORStreatment\was administered, there were significant levels of NOdetectable by AeroNOx™ (Pulmonox, Canada) device. These results showthat the bioavailability of NO produced from NORS was demonstrated inthe serum after treatment.

The average temperature for control versus treated animals, after 3 and5 days was significantly (P<0.05) higher (FIG. 18B). This shows asystemic effect of the NORS treatment on the ferrets.

The invention claimed is:
 1. A liquid nitric oxide releasing solution(NORS) comprising at least one nitric oxide releasing compound and atleast one acidifying agent, wherein the NORS provides an extendedrelease of a therapeutically effective amount of nitric oxide gas (gNO),wherein the gNO is released over a period of at least 30 minutes, andwherein either the amount of the at least one nitric oxide releasingcompound is not greater than about 0.5% w/v, or the amount of the atleast one acidifying agent is not greater than about 0.5% w/v, andwherein the NORS has a pH of from about 1 to about 4 during gNO release.2. The solution of claim 1, wherein the at least one nitric oxidereleasing compound is selected from the group consisting of a nitrite, asalt thereof, and any combinations thereof.
 3. The solution of claim 2,wherein the nitrite is sodium nitrite.
 4. The solution of claim 1,wherein the amount of the at least one nitric oxide releasing compoundis not greater than about 0.5% w/v.
 5. The solution of claim 1, whereinthe at least one acidifying agent is an acid.
 6. The solution of claim5, wherein the acid is citric acid.
 7. The solution of claim 1, whereinthe amount of the at least one acidifying agent is no greater than about0.5% w/v.
 8. The solution of claim 1, wherein the therapeuticallyeffective concentration of NO is about 160 ppm.
 9. The solution of claim1, wherein the NORS is a saline-based solution.
 10. The solution ofclaim 1, wherein the gNO is released over a period of at least 4 hours.11. The solution of claim 1, wherein the gNO is released over a periodof at least 8 hours.
 12. The solution of claim 1, wherein the gNO isreleased over a period of at least 12 hours.
 13. The solution of claim1, wherein the gNO is released over a period of at least 24 hours.